cDNA cloning and endogenous ligand identification of the novel ligand-gated ion-channel receptors

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5HT3C is the new member of serotonin-gated ionotropic receptor. From the sequence homology search for 5HT3C cDNA against human genomic database, three additional receptor genes, designated 5HT3D, 3E and 3L1, were identified on the same chromosome 3q27.1. These four predicted genes are composed of nine exons and share common features in exon-intron organization with previously known serotonin receptors 5HT3A and 3B. RT-PCR analysis in human tissues demonstrated that 5HT3C mRNA was highly expressed in placenta, lung and kidney, whereas 5HT3D mRNA was seen in lung and pancreas. The cDNA cloning revealed that alternative splicing form lacking exon 2,3,4 and 6 of 5HT3C and one lacking exon 3 of 5HT3D were expressed in kidney and pancreas, respectively. To compare the expression levels of these mRNA in each tissue, PCR-RFLP assay was performed by using common primers designed from the conserved sequences in exon 7 and exon 9. In brain, lung and liver, 5HT3C transcript was dominant, while 5HT3D message was mainly expressed in heart. Coexpression of 5HT3D and 5HT3L1 was demonstrated in skeletal muscle while 5HT3C, 5HT3D and 5HT3L1 were co-expressed in placenta, kidney and pancreas. The expression of 5HT3E transcript could not be detected, and hence, this gene was assumed as a pseudogene. Furthermore, other splicing variants at the junction of exon 7 and exon 8 of 5HT3D and 5HT3L1 were expressed in pancreas. Taken together, these results suggest that novel identified ion-channel receptors execute or modulate many physiological functions in diverse tissues through oligomerization of various splicing variants.